The central theme of this program project is conceptual. Specifically, we propose that limb morphogenesis proceeds from intrinsic mechanisms within mesenchyme which can be extrinsically modulated by morphogens secreted by adjacent tissues. We believe to elucidate these mechanisms, it is critical that specific endogenous limb molecules be identified that coordinate expression of the various limb phenotypes. We especially seek to identify proteins that serve to establish the primary pattern of tissue organization upon which limb morphogenesis proceeds. High resolution molecular approaches will be used to structurally and functionally characterize specific proteins (some already in hand) for their effects on lineage restriction, phenotypic expression and tissue formation of limb mesenchymal cells. All projects utilize a 3.d culture system in which subpopulations of mesenchyme can be isolated or probed for response to histogenic inductive signals. All projects include either molecular cloning or chromatin structural analyses. For each projects include either molecular cloning or chromatin structural analyses. For each project, specific hypotheses have been proposed. In Project I (Markwald/Krug/Bolender) ectodermally secreted proteins (e.g. ES-I antigens) will be assayed for effects for their regulatory influence on chondrogenesis (including regulation of the number of primary chondrogenic centers) nd histogenic interactions between mesenchymal precursor cell populations in collagen gel culture. In Project III (Godfrey) the role of basement membrane molecules found on mesenchymal cells before cytodifferentiation will be functionally tested for their role(s) in myogenic differentiation, myotube formation and synaptogenesis. For one of these, agrin, cDNA clones will be obtained for studies of developmental regulation including ectodermal interaction. In Project IV (Fallon), in vivo analyses will be performed to test the roles of ectoderm in patterning mechanisms while the 3d collagen gel culture will be used to establish bioassays of apical ectodermal ridge function using limbless (11) and wingless mutants. Results from both Projects I and IV will compared with findings obtained from the pattern deficient Talpid 2 mutant. Dr. Fallon and his collaborator, Dr. Temin, will continue their ongoing studies to develop a chick heritable marker for use in cell marking experiments in Projects I and IV. Three core units, Primary Culture Core (Bolender), Monoclonal Antibody Core (Yorde) and the EM Cytochemical Core (Riley) along with an Administrative Core will provide the research projects with both technical and conceptual support. Enthusiasm for the program remains high as progress has resulted in the development of procedures and molecular probes with which we can pursue interdisciplinary efforts to elucidate mechanisms of limb morphogenesis.